Tissue culture device



June 28, 1960 s. G. ROSE 2,942,520 TISSUE CULTURE msvxcs Filed Dec. 20,1955 Geo/ye 6. Rome INVENTOR.

pwfliw f ATTORNEYJ United States Patet TISSUE CULTURE DEVICE George G.Rose, 114 McTighe Drive, Bellaire, Tex.

Filed Dec. 20, 1955, Ser. No. 554,218

2 Claims. (Cl. 88-40) This invention relates to a tissue culture deviceand more particularly to one which may be used intact with microscopesand especially phase contrast microscopes.

Most tissue culture chambers are not as suitable as desired formicroscopic examination of the contents of the chambers while thecontents are still in the chambers and most microscopic slides have notbeen easily adapted to good tissue culture chambers. Several devicesattempt to combine both the attributes of a good tissue culture chamberand a good microscopic slide but none of these devices fully accomplishthis aim.

It is a general object of the present invention to provide a tissueculture device including a chamber which device after its usefulness fora particular culture may be dismantled and the cells fixed, stained andthe slide mounted for a permanent record; which permits microscopicexamination of any magnitude including oil magnitude at any desired timewithout altering the device or environment of the cells; which can beperfused with known or unknown fluids and its nutrient exchanged atwill; which possesses good aseptic technical qualities; which containsadequate surface for extended culturing; which uses relatively smallamount of nutrient, which does not need plasma clots; which permitsvital staining (staining of living tissue) without disrupting thecellular growth or altering the structure of the device; and whichprovides a system of cultivation conducive to cultural survival overlong periods of time.

Another object of the present invention isto provide such a tissueculture device permitting the use of hypodermic needles for theextraction and'insertion of nutrients and other matter.

And still a further object of the present invention is to provide such atissue culture device which is reliable, durable, and easy to handle.

Other objects and advantages will be more apparent from the followingdescription of a preferred example of the invention, given for thepurpose of disclosure, and taken in conjunction with the accompanyingdrawings, where like character references refer to like parts throughoutthe several views and where Fig. l is a perspective view of the tissueculture device of the present invention showing a hypodermic syringe andneedle for insertion or withdrawal of matter from the chamber and ahypodermic needle for maintaining constant pressure in the chamber,

Fig. 2 is an exploded view of'the tissue culture device inverted fromthe position shown in Fig. 1, and

Fig. 3 is a view along line 3-3 of Fig. 1.

Referring now to the drawings, and particularly to Fig. 2, the tissueculture device as a whole includes first and second retaining plates 12and 14 respectively each having a transparent cover slip 16 and 18adjacent its in terior side and a gasket 20 between the cover slips 16and 18, all held together in a sandwich by bolts 22. Apertures 24 and 26are formed in the first and second retaining plates 12 and 14respectively and are in mutual alignment with one another so that whenthe tissue culture "ice device is'assembled as shown in Figs. 1 and 3thereis a chamber '28 formed by an aperture 30 in the gasket extendedculture growth without increasing the dimensions of the cover plates tosuch a size that they are not 7 convient for handling these cover plates12 and 14 need to' be made of a rigid material. Stainless steel platesmeasuring 2" x 3" x A" have been found very satis factory. Stainlesssteel has the desired qualities of rigidity and is readily available.The 2" x 3" length and width is convenient to handle and allows acomparatively large aperture 24 or 26 to be formed while using retainingplates 12 and 140i only A; in thickness.-

With the particular dimensions of the retaining plates 12 and 14previously given an aperture of 1 in diameter centered in each plate 12and 14 may be used which is of ample size for most extended culturegrowth and yetwill leave sufiicient material in the retaining plates 12and 14 to prevent harmful bowing.

As the lower end of the objectives of some microscopes, V

V and especially of phase contrast microscopes,needs to be placed inclose proximity to the specimen, and as some of the tissue to beexamined by the microscope may be at the periphery of the apertures, theaperture 24 in the first retaining plate 12, which is the one to beplaced adjacent the objective of the microscope, is countersunk as at 32(see Fig. 3) so that the objective may be placed close to the cover slip16 at the periphery of aper-- ture 24.

The cover slips 16 and 18 are normally identical and I the gasketmaterial.

ness which is from 0.13 to 0.17 millimeter in thickness. Such coverslips are thin enough to permit the objective of a microscope to be inclose proximity to a tissue culture specimen lying on the underneathside of the cover 1 I slip 16 or 18 adjacent themicroscope. Thesestandard optical cover slips 16' and 18 of No. l thickness are; marketedin various-sizes and one standard size is 50 x' 43 millimeters which isapproximately the same. si'z efas the.

gasket 20 and therefore such size cover slip is desirable.

The gasket 20 makes a; sealing engagement witn the cover slips 16 and 18to form the chamber, 23 ari' d 'it also permits introduction andwithdrawal at"willof needles such' as the hypodermic needle's '34 and36'best seen in Fig. l. This gasket 20 must be non-toxic to theparticular cultures in thechamber 2 8, needle penetratable, andself-sealing so that when hypodermic needles are withdrawnthere is nopassage of fluid to or from the chamber 28. Further, this gasket 20 mustbe of material that when placed in the sandwich will form a fluid tightseal with the cover slips 16 and 18. Pure gum latex has been found tohave all these properties and is highly satisfactory; However, for somereason certain pure gum latex gaskets are .toxic to varying extents uponmany tissue cultures and care must be used in the choice of 18 and thusto help prevent bowing and additionally to provide a maximum sealingsurface between the gasket 20 and the cover slips 16 and 18, theaperture 30 in'gasket 20 is preferably made the same diameter as theapertures 24 and 26 in retaining plates 12 and 14 respectively.

Patented June 28, 1960 Pure gum latex (floating stock) With theretaining plates 12 and 14 of the dimensions previously given a gasket20 measuring 2" x 1%" has been found to be very satisfactory as it hassufiicient size toform a seal after a needle is withdrawn and hassufficient surface contacting the cover slips ioand 13 to preventslippage when the needle is inserted'or withdrawn. Using cover slips ofNo; 1 thickness and retaining plates thick, a gasket 20 approximatelythick is highly satisfactory in that the gasket is sufficiently thick topermit hypodermic needles to be easily insertedand withdrawn and yet thedistance between the underneath side of the cover slip placed adjacentthe objective of the microscope (the tissue culture specimen is usuallylocated here) and the outer surface of the retaining plate nearest thecondenser is no greater than the maximum working distance permitted bymost phase contrast microscopes (having long working distancecondensers) between the condenser of the microscope and the specimen tobe examined, even in oil magnification study.

To hold the tissue culture device assembled in the position shown inPig. 1, four corner holes 40 (see Fig. 2) are provided in one of theretaining plates such as the second retaining plates 14 and the holes 46are counter sunk to receive the Allen bolts 22 so that the heads of theAllen bolts 22 do not project above the surface of the retaining plate14. The other retaining plate, 12 in this instance, has internallythreaded holes 38 which are complementary with the holes 40 in thesecond retaining plate and in which the Allen bolts 22 may be threadedlyengaged Without extending beyond the outer surface of retaining plate12.

The first and second retaining plates 12. and 14 need to be heldtogether by means on opposite sides of the chamber 23 so that force orithe cover slips 16 and 18 may be adjusted to prevent bowing of thesecover slips and to adjust other stresses on them which affect theiroptical qualities. Additionally, the means to secure the retainingplates 12 and 14 to one another should not extend beyond the outermostsurface of these retaining plates as any such projection would preventuse of the device with certain microscopes and would make itinconvenient in storage and in handling generally. These four Allenbolts 22 located as illustrated have been found to be satisfactory.

When the tissue culture device is being assembled the hypodermicneedle34, used as a vent, should be inserted through the gasket 20, asiilustratedin Fig. 1, before a final tightening of the Allen bolts 22takes place as'pressure build-up in ti.e chamber 28 during the finaltightening sometimes ruptures cover slips 16'and 18. This vent needle34may beleft in place with a cotton plug in it as convenientbacteriological filter or it may be withdrawn. fluids may be introducedinto the chamber 28 or withdrawn from it by using the hypodermic needle36 and syringe 42 but whenever this is done the vent needle 34 shouldalso be used to prevent pressure changes within the chamber 28.

With this particular device entries and withdrawals of the hypodermicneedle may be made at will without the introduction of any contaminationinto the chamber, thus providing a device which may be perfused or haveits nutrient changed at will and possessing good aseptic qualities.Further, because the nutrient may be changed at Once the tissue culturedevice is assembled.

will plasma clots, which are often objectionable in microscopic studies,are not necessary. Additionally, vital staining may take place Withoutdisrupting the cellular growth by introduction of the dyes directly intothe chambar from the hypodermic needle 36 and syringe 42. Further, thedyes may be washed out of the culture and chamber by the same method. ifstaining of the culture 1. is desired this can be done on the coverslips 16 and 18 with the cover slips being mounted as a permanentrecord.

The tissues to be cultured may be introduced into the chamber 28 ineither of two ways. The tissue culture device may be assembled asillustrated in Fig. 1 and the tissue introduced by the hypodermic needle36 and syringe 42, or, if the fragments are too large or plasma clotsare desired, then the tissue is placed within the chamber 28 before thetop cover slip is positioned and thereafter the device is assembled asillustrated in Fig. l.

All parts of the tissue culture chamber are re-usable except one of thecover slips whenever staining is desired or for some reason it isdesired to keep the culture on that particular cover slip. Thereforemaintenance cost is low.

Although dimensions have been given, it is to be understood that theseare not critical but are given for the purpose of illustration only.

While only a single example of the invention has been given for thepurpose of illustration, changes in detail will suggest themselves tothose skilled in the art. Accordingly, it is desired to be limited onlyby the spirit of the invention as defined by the scope of the appendedclaims.

What is claimed is:

l. A tissue culture device comprising, first and second rigid retainingplates in parallel spaced relationship each having an aperturetherethrough in mutual alignment, a gum latex gasket between theretaining plates with its outer edges exposed, said gasket having anaperture therethrough aligned with the other apertures and said gumlatex gasket being nontoxic to tissue to be used, transparent coverslips between the gasket and the adjacent retaining plates, said coverslips being in mutual alignment over the aperture in the gasket and eachcover slip contacting a retaining plate, one of said plates havingthreaded openings on opposite sides of the aperture, and

threaded means connected to the other retaining plate on opposite sidesof the aperture and threadable in the threaded openings therebyreleasably and adjustably holding the retaining plates, cover slips, andgasket sealingly together whereby said assembled culture device isadapted to be placed under a microscope for observance of the tissueculture therein.

2. The tissue culture device of claim 1 in. which the cover slips are ofstandard No. 1 optical thickness.

References Cited in the file of this patent UNITED STATES PATENTS504,890 Ohmart Sept. 12, 1893 681,400 McCarty Aug. 27; 1901 2,048,128Logan July 21, 1936 2,144,255 Carpenter Jan. 17, 1939 2,348,448 BrewerMay 9, 1944 2,644,452 Brown July 7, 1953 FOREIGN PATENTS 936,299 GermanyDec. 7, 1955

